PKC-Theta-Mediated Signal Delivery from Thetcr/CD28 Surface Receptors

REVIEW ARTICLE published: 22 August 2012 doi: 10.3389/ﬁmmu.2012.00273 PKC-theta-mediated signal delivery from theTCR/CD28 surface receptors

Noah Isakov1* and Amnon Altman2 1 The Shraga Segal Department of Microbiology and Immunology, Faculty of Health Sciences and the Cancer Research Center, Ben-Gurion University of the Negev, Beer Sheva, Israel 2 Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Edited by: Protein kinase C-theta (PKCθ) is a key enzyme inT lymphocytes, where it plays an important Nick Gascoigne, Scripps Research role in signal transduction downstream of the activated T cell antigen receptor (TCR) and Institute, USA the CD28 costimulatory receptor. Interest in PKCθ as a potential drug target has increased Reviewed by: following recent findings that PKCθ is essential for harmful inflammatory responses medi- Balbino Alarcon, Consejo Superior de Investigaciones Cientificas, Spain ated byTh2 (allergies) andTh17 (autoimmunity) cells as well as for graft-versus-host disease Salvatore Valitutti, INSERM, France (GvHD) and allograft rejection, but is dispensable for beneficial responses such as antiviral *Correspondence: immunity and graft-versus-leukemia (GvL) response. TCR/CD28 engagement triggers the Noah Isakov, The Shraga Segal translocation of the cytosolic PKCθ to the plasma membrane (PM), where it localizes at Department of Microbiology and the center of the immunological synapse (IS), which forms at the contact site between an Immunology, Faculty of Health Sciences and the Cancer Research antigen-specificT cell and antigen-presenting cells (APC). However, the molecular basis for Center, Ben-Gurion University this unique localization, and whether it is required for its proper function have remained of the Negev, P.O. Box 653, unresolved issues until recently. Our recent study resolved these questions by demon- Beer Sheva 84105, Israel. θ e-mail: [email protected] strating that the unique V3 (hinge) domain of PKC and, more specifically, a proline-rich motif within this domain, is essential and sufficient for its localization at the IS, where it is anchored to the cytoplasmic tail of CD28 via an indirect mechanism involving Lck protein tyrosine kinase (PTK) as an intermediate. Importantly, the association of PKCθ with CD28 is essential not only for IS localization, but also for PKCθ-mediated activation of downstream signaling pathways, including the transcription factors NF-κB and NF-AT,which are essential for productive T cell activation. Hence, interference with formation of the PKCθ-Lck-CD28 complex provides a promising basis for the design of novel, clinically useful allosteric PKCθ inhibitors. An additional recent study demonstrated that TCR triggering activates the germinal center kinase (GSK)-like kinase (GLK) and induces its association with the SLP-76 adaptor at the IS, where GLK phosphorylates the activation loop of PKCθ, converting it into an active enzyme. This recent progress, coupled with the need to study the biology of PKCθ in human T cells, is likely to facilitate the development of PKCθ-based therapeutic modalities for T cell-mediated diseases.

Keywords: protein kinase C-theta, PKCθ, CD28, Lck, signal transduction, costimulation

INTRODUCTION process and describes in more detail the studies that clarified a new Protein kinase C-theta (PKCθ) is a key regulator of signal trans- mechanism by which PKCθ is being recruited to the center of the duction in activated T cells that is linked to multiple pathways IS and is essential for the induction of PKCθ-dependent activation downstream of the T cell antigen receptor (TCR; Isakov and signals. Altman, 2002). Engagement of the TCR and the resulting formation of diacylglycerol (DAG) are sufficient for promoting PKCθ THE PKC FAMILY recruitment to cell membranes (Monks et al., 1997, 1998). How- Protein kinase C was discovered by Nishizuka and colleagues, who ever, localization of PKCθ to the immunological synapse (IS) is demonstrated a new kinase that undergoes activation by lim- entirely dependent on the concomitant ligation of the CD28 core- ited proteolysis (Inoue et al., 1977), or by translocation to the ceptor (Huang et al., 2002). Localization of PKCθ at the center plasma membrane (PM), where it associates with specific cofac- of the IS is essential for activation of signaling pathways that tors (Takai et al., 1979). The membrane-associated PKC-activating promote T cell-dependent immune responses against distinct anti- factor turned to be DAG (Kishimoto et al., 1980). DAG, together gens and pathogens. While the recruitment of PKCθ to the IS of with inositol 1,4,5-trisphophate (IP3), are products of phospho- TCR/CD28 engaged T cells has been extensively studied, informa- lipase C-mediated hydrolysis of the membrane phospholipid, tion on the molecular basis for this highly selective process has phosphatidylinositol 4,5-bisphosphate (PIP2; Berridge and Irvine, been relatively scarce until recently. The present manuscript pro- 1984; Nishizuka, 1984). These two second messengers transduce vides background information on the molecules involved in this signals from a plethora of activated receptors: the hydrophobic

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DAG remains bound to the cell membrane where, in addition to events leading to activation of transcription factors, including PKC, it activates effector molecules such as RasGRP, a guanine NF-κB, AP-1, and NF-AT, which are critical for T cell activa- nucleotide exchange factor (GEF) for Ras (Lorenzo et al., 2000), tion, proliferation and differentiation (Baier-Bitterlich et al., 1996; while the hydrophilic IP3 diffuses through the cytosol and binds Coudronniere et al., 2000; Dienz et al., 2000; Lin et al., 2000; 2+ IP3-receptors, which function as ligand-gated Ca channels in Sun et al., 2000; Pfeifhofer et al., 2003). Under certain activa- the endoplasmic reticulum (ER), thereby triggering the release of tion conditions, PKCθ can translocate to the nucleus where it free Ca2+ ions into the cytoplasm (Takai et al., 1979; Khan et al., directly associates with chromatin and is involved in the regu- 1992; Bourguignon et al., 1994). The utilization of phorbol esters, lation of microRNAs and T cell-specific inducible gene expression which mimic the activity of DAG, together with Ca2+ ionophores, program (Sutcliffe et al., 2011). The exact mechanism by which demonstrated that PKC also plays an essential role in the induc- the membrane-bound PKCθ delivers signals to the nucleus has tion of T lymphocyte proliferation (Truneh et al., 1985; Isakov and not been fully resolved but studies provided information on a Altman, 1987) and reactivation of effector cytotoxic T cells (Isakov number of effector molecules that operate along this pathway and Altman, 1985; Isakov et al., 1987). in activated T cells. These studies demonstrated that PKCθ- Protein kinase C enzymes transduce a myriad of signals from mediated regulation of NF-κB activity involves the multisubunit a large number of cell surface receptors that are coupled to inhibitor of κB(IκB) kinase (IKK) complex (Coudronniere et al., phospholipase C and phospholipid hydrolysis. They regulate the 2000; Dienz et al., 2000; Khoshnan et al., 2000; Lin et al., 2000; function of effector molecules by phosphorylating specific serine Bauer et al., 2001). and threonine residues. The PKC family includes 10 structurally An important upstream effector in the NF-κB signaling path- and functionally related isoforms (for more details, see the first wayisIκBα, which binds NF-κB in the cytoplasm of resting T cells review by Pfeifhofer-Obermair et al., 2012), grouped into three and mask its nuclear localization signal (NLS), thereby preventing subfamilies based on the composition of their regulatory domains NF-κB translocation to the nucleus (Mercurio et al., 1997; Regnier and their respective cofactor requirements (Newton, 1995; Mel- et al., 1997; Jacobs and Harrison, 1998). IKK-mediated phospho- lor and Parker, 1998). The first subfamily includes conventional rylation of IκBα signals the protein for degradation (Karin, 1999), PKCs (cPKC; α, βI, βII, γ) that are regulated via two DAG-binding exposes the NF-κB NLS and promotes NF-κB translocation to C1 domains organized in tandem near the cPKC amino termi- the nucleus and the induction of NF-κB-mediated gene transcrip- nus (Hurley et al., 1997; Johnson et al., 2000; Ho et al., 2001) tion.TcellsfromPKCθ-deficient (Prkcq−/−) mice fail to respond and an adjacent Ca2+ and phospholipid-binding C2 domain to TCR stimulation with degradation of IκBα (Sun et al., 2000), (Nalefski and Falke, 1996; Johnson et al., 2000). The second supporting the model whereby PKCθ regulates NF-κB activity group includes novel PKCs (nPKC; δ, ε, η, θ) that are DAG- through its effect on IKK-IκBα. Some of the effector molecules dependent, but Ca2+ and phospholipid independent for their that link PKCθ to IKK have been identified and include the PKCθ activity. The third group includes atypical PKCs (aPKC: ζ, λ/ι) substrate protein, caspase activation and recruitment domain that are DAG-, Ca2+-, and phospholipid-independent. While (CARD) and membrane-associated guanylate kinase (MAGUK) PKC enzymes are involved in metabolic processes in different domain-containing protein-1 (CARMA1). This scaffold protein cell types, many studies implicate PKC enzymes in signal trans- is primarily expressed in lymphocytes (Bertin et al., 2001; Hara duction networks that convert environmental cues into cellular et al., 2003), where it links PKCθ to NF-κB activation in T cells actions (Rosse et al., 2010). Six of the PKC isoforms, includ- (Ruland et al., 2001, 2003; Ruefli-Brasse et al., 2003; Xue et al., ing PKCα, δ, ε, η, θ, and ζ are expressed at varying amounts 2003). Phosphorylation of CARMA1 by PKCθ in TCR/CD28- in T cells (Meller et al., 1999). Immunological studies using dif- stimulated T cells, promotes CARMA1 association with the B-cell ferent genetic models and pharmacological drugs indicated that lymphoma/leukemia 10 (Bcl10) and mucosa-associated lymphoid distinct PKC isoforms are required for different aspects of the tissue 1 (MALT1) proteins (Matsumoto et al., 2005; Sommer et al., activation and effector functions of T cells. The results sug- 2005) leading to recruitment of the trimolecular complex to the gest that distinct PKC isoforms may serve as drug targets for IS (Gaide et al., 2002; Che et al., 2004; Hara et al., 2004) and different T cell mediated adaptive immune responses (Baier and activation of the IKK complex (McAllister-Lucas et al., 2001). Fur- Wagner, 2009). thermore, overexpression of CARMA1, Bcl10, and MALT1 in T cells, followed by TCR/CD28 stimulation, resulted in the forma- PROTEIN KINASE C-THETA tion of a CARMA1-Bcl10-MALT1 trimolecular complex, where Protein kinase C-theta is a Ca2+-independent nPKC isoform all three proteins were required for maximal activation of NF-κB exhibiting a relatively selective pattern of tissue distribution, with (McAllister-Lucas et al., 2001; Ruland et al., 2001). It should be predominant expression in T lymphocytes (Baier et al., 1993; noted that in some studies (Khoshnan et al., 2000), but not oth- Meller et al., 1999), platelets (Chang et al., 1993; Meller et al., ers (Lin et al., 2000), PKCθ was found to directly associate with 1998; Cohen et al., 2009), and skeletal muscle (Osada et al., 1992; members of the IKK complex, particularly IKKβ, suggesting the Chang et al., 1993). It has a unique ability to translocate to potential existence of an additional linear route from PKCθ to the center of the IS of activated T cells (Monks et al., 1997, NF-κB. The transcription factor AP-1, similar to NF-κB,isapri- 1998) where its full activation requires the integration of TCR mary physiological target of PKCθ (Baier-Bitterlich et al., 1996; Li and CD28 costimulatory signals (Huang et al., 2002; Tseng et al., et al., 2004), while regulation of the NF-AT transcription factor 2008; Yokosuka et al., 2008). Engagement of the TCR and the requires cooperation between PKCθ and calcineurin, a Ca2+- CD28 coreceptor initiates a series of PKCθ-dependent signaling dependent serine/threonine phosphatase (Pfeifhofer et al., 2003).

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All three PKCθ-regulated transcription factors have correspond- model. In these studies, Rag−/− mice reconstituted with Prkcq−/− ing binding sites on the IL-2 gene promoter, and their bind- T cells were unable to reject cardiac allografts, in contrast to the ing to the IL-2 gene is essential for optimal IL-2 response acute allograft rejection observed in the wild-type T cell recon- (Isakov and Altman, 2002). stituted Rag−/− mice. However, this was due to lack of PKCθ- While PKCθ-mediated regulation of NF-κB activity in regulated expression of anti-apoptotic molecules, such as Bcl-xL, TCR/CD28-stimulated T cells has been studied in great detail, which led to apoptosis of the effector T cells; transgenic expression −/− PKCθ is also involved in the regulation of additional cellular func- of Bcl-xL in Prkcq T cells restored their ability to reject the car- tions, and physically associates with additional binding partners. diac allografts. The rejection of cardiac allograft by Prkcq−/− mice Besides CARMA1, PKCθ physically associate with 14-3-3τ (Meller was only slightly delayed (Manicassamy et al., 2008; Gruber et al., et al., 1996), Cbl (Liu et al., 1999), Fyn (Ron et al., 1999), Lck (Liu 2009), suggesting compensation by other PKC isoforms. Indeed, et al., 2000), AKT (Bauer et al., 2001), moesin (Pietromonaco et al., mice lacking both PKCθ and PKCα, demonstrated a significantly 1998), PICOT (Witte et al., 2000), and the HIV nef protein (Smith delayed rejection of cardiac allografts (Gruber et al., 2009). et al., 1996). Some of these molecules (i.e., Lck) phosphorylate The overall positive role of PKCθ in the activation of effector PKCθ and may affect its activity and/or subcellular distribution, Tcells(Teff ) and the promotion of adaptive immune responses while others, which serve as substrates for PKCθ (i.e., Cbl, 14-3-3τ raise questions about the nature of its function in regulatory T and moesin) may regulate cellular functions, such as cytoskeletal cells (Treg) that suppress Teff functions. This issue has recently reorganization. been partially resolved by Zanin-Zhorov et al. (2010) who found that PKCθ mediates negative feedback on Treg functions. Further- θ DIFFERENTIAL REQUIREMENTS FOR PKC BY more, activation of Treg resulted in sequestration of PKCθ away DISTINCT T CELL SUBPOPULATIONS from the IS, and inhibition of PKCθ activity (using the C20 com- Initial characterization of PKCθ-deficient T cells suggested the pound) increased the suppressive activity of Treg (Zanin-Zhorov involvement of PKCθ in cellular responses leading to T cell acti- et al., 2010, 2011). In vivo studies demonstrated that Treg devel- vation, proliferation, and cytokine production (Sun et al., 2000; opment in the thymus of Prkcq−/− mice is impaired leading Pfeifhofer et al., 2003; Anderson et al., 2006). Subsequent in vitro to reduced numbers of Treg cellsintheperiphery(Schmidt- and in vivo investigations and the analysis of Prkcq−/− mice in dif- Supprian et al., 2004; Zanin-Zhorov et al., 2010, 2011), although ferent disease models demonstrated differential requirements for activity of these mature PKCθ-deficient Treg cells was intact PKCθ by distinct T cell subpopulations and during the induc- (Gupta et al., 2008). tion of selected types of immune responses. Thus, PKCθ was found to be essential for the induction of Th2-type immune THE IMMUNOLOGICAL SYNAPSE responses to allergens or helminth infection (Marsland et al., 2004; Adaptive immune responses are dependent on the effective com- Salek-Ardakani et al., 2004) and the induction of Th17-mediated munication between antigen-specific T cells and APCs. At the experimental autoimmune encephalomyelitis (EAE) that serves very early phase of the activation response, T cells interact via as a model of multiple sclerosis (Salek-Ardakani et al., 2005; their TCR with cognate peptide-MHC complexes on the surface Anderson et al., 2006; Tan et al., 2006; Marsland et al., 2007; of APCs and both cell types respond by redistributing their recep- Kwon et al., 2012), and other experimental autoimmune dis- tors/ligands to the contact area that rearranges as a platform for eases (Anderson et al., 2006; Healy et al., 2006; Marsland et al., effective signaling (Dustin and Zhu, 2006). The IS, representing 2007; Chuang et al., 2011). In contrast, Th1-dependent mouse the interface between a T cell and an APC, is formed by specific resistance to Leishmania major infection was intact in Prkcq−/− protein microclustering (Yokosuka et al., 2005) and their segre- mice (Marsland et al., 2004; Ohayon et al., 2007), and PKCθ gation into one of two separate regions: a central core [central was dispensable for CTL-mediated protective antiviral responses, supramolecular activation clusters (cSMAC)], which contains the most likely reflecting compensation by innate immunity sig- TCR and costimulatory receptors, and a peripheral region [periph- nals (Berg-Brown et al., 2004; Giannoni et al., 2005; Marsland eral supramolecular activation clusters (pSMAC)], which contains et al., 2005, 2007; Valenzuela et al., 2009). Consistent with the adhesion molecules, such as LFA-1 (Dustin, 2009). T cell sur- in vivo findings, in vitro induction of CD4+ T cell polariza- face receptor engagement triggers signaling cascades that result in tion by optimal T cell-antigen-presenting cell (APC) coculture the recruitment of multiple membrane-anchored and cytoplas- conditions, demonstrated a requirement for PKCθ during Th2 mic effector molecules, including kinases, adaptor proteins, and and Th17 cell development, and only moderate effect of PKCθ cytoskeletal components, to the IS (Dustin et al., 2010). One of on Th1 cell development (Marsland et al., 2004; Salek-Ardakani the most prominent proteins to be recruited to the IS of antigen- et al., 2004, 2005). Additional studies performed in Prkcq−/− responding T cells is PKCθ, which localizes at the cSMAC (Monks mice demonstrated the requirement for PKCθ in the induction of et al., 1997, 1998). Additional high-resolution imaging analysis graft-versus-host (GvH) and alloreactive T cell-mediated immune by TIRF microscopy demonstrated that PKCθ colocalizes with responses (Valenzuela et al., 2009). In contrast, PKCθ-deficient T CD28, and demonstrated that the cSMAC is divided into two cells retained the ability to induce graft-versus-leukemia (GvL) structurally and functionally distinct compartments: a central responses in allogeneic bone marrow (BM) transplanted mice TCRhigh compartment, where signaling is terminated (Vardhana (Valenzuela et al., 2009). et al., 2010) and TCR-associated signaling complexes are internal- Protein kinase C-theta also contributes to allograft rejection, ized and degraded, and an outer TCRlow “ring” where PKCθ and as shown by Manicassamy et al. (2008) using an adoptive transfer CD28 colocalize (Yokosuka et al., 2008).

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CD28 SIGNALING DOWNSTREAM OF CD28 CD28 is a type 1 transmembrane glycoprotein that is constitu- CD28 delivers signals in activated T cells via its cytoplasmic tively expressed as a disulfide-linked homodimer on all CD4+ and tail, which has no intrinsic catalytic activity, but possesses sev- CD8+ murine T cells and majority of CD4+ and CD8+ human eral protein–protein interaction motifs that enable it to associate peripheral blood T cells (Gross et al., 1990; Vallejo, 2005). The with enzymes and other effector molecules (Boise et al., 1993;see human CD28 precursor protein is 220 amino acids long (218 in Figure 1). In resting T cells, non-phosphorylated CD28 asso- mouse) and the mature protein possesses 202 amino acids (218 ciates with the serine/threonine protein phosphatase protein 2A in mouse) due to cleavage of an amino-terminal leader sequence (PP2A), which dissociates from CD28 upon activation induced- (18 and 19 amino acids in the human and mouse CD28, respec- phosphorylation of CD28 (Chuang et al., 2000). CD28 triggering tively). In addition, CD28 possesses a cytoplasmic tail of 41 amino by its ligands leads to phosphorylation of tyrosine residues (Raab acids (38 in mouse) that is critical for signal transduction and et al., 1995; Teng et al., 1996; King et al., 1997) in the cytoplas- coreceptor-induced cell stimulation. Physiological activation of mic tail of CD28, creating new docking sites for different effector CD28 is mediated by one of two natural ligands expressed on molecules that initiate the activation of signaling cascades, and the surface of APCs, CD80, and CD86, which directly asso- define the costimulatory functions of CD28 (Raab et al., 1995; ciate with a conserved motif [MYPPPY (single amino-acid letter Andres et al., 2004; Dodson et al., 2009). code)] in the extracellular region of CD28 (Kariv et al., 1996; The first motif in the human CD28 cytoplasmic tail, juxta- Truneh et al., 1996). Engagement of CD28 provides costimula- posed to the PM, contains a Y173MNM sequence that undergoes tory signals that complement or synergize with those provided tyrosine phosphorylation following the engagement of CD28 and by the TCR, leading to optimal activation of T cells (Thompson serves as a binding site for the SH2 domain of p85, the regulatory et al., 1989; Harding et al., 1992). CD28 engagement increases IL- subunit of the lipid kinase, phosphatidylinositol 3-kinase (PI3K; 2production(Thompson et al., 1989; Jain et al., 1995; Reichert August and Dupont, 1994; Pages et al., 1994; Prasad et al., 1994; et al., 2001) and IL-2 receptor expression (Shahinian et al., 1993), Truitt et al., 1994). The methionine residue at the +3 position and provides survival signals by upregulating the anti-apoptotic confers specificity for p85 binding (Takeda et al., 2008), while the protein, Bcl-XL (Boise et al., 1993). In addition, CD28 syner- asparagine at the +2 position confers additional specificity for gizes with the TCR in providing potent signals for activation of the SH2 domain of Grb2 and GADS (Songyang et al., 1993; Raab c-Jun kinase (JNK), p38 MAP kinase, and IKK pathways (Su et al., 1995; Sanchez-Lockhart et al., 2004; Schneider et al., 1995; et al., 1994; Harhaj and Sun, 1998), and activation of the NF- Harada et al., 2001). The relative concentration of PI3K, Grb2, and κB(Michel et al., 2000; Diehn et al., 2002)AP-1(Rincon and GADS at the vicinity of CD28 cytoplasmic tail, and the relative Flavell, 1994) and NF-AT transcription factors (Michel et al., 2000; affinity of their SH2 domain for the phospho-Tyr173-containing Diehn et al., 2002). The positive role of CD28 in T cell activation was demonstrated in CD28-deficient (Cd28−/−) T cells, in which TCR engagement in the absence of CD28 costimulation resulted in anergy and/or tolerance induction upon rechallenge with the same antigen (Appleman and Boussiotis, 2003). T cell proliferation and Th2- type cytokine secretion were also severely impaired in Cd28−/− mice or wild-type mice treated with CD28 antagonists (Green et al., 1994; Lucas et al., 1995; Rulifson et al., 1997; Schweitzer et al., 1997; Gudmundsdottir et al., 1999). Furthermore, lack of CD28-mediated costimulation led to reduced immune responses against infectious pathogens (Shahinian et al., 1993; King et al., 1996; Mittrucker et al., 2001; Compton and Farrell, 2002) and allografts (Salomon and Bluestone, 2001) and impaired GvH disease (Via et al., 1996), contact hypersensitivity (Kondo et al., 1996), and asthma (Krinzman et al., 1996). FIGURE 1 | Signaling motifs in the cytoplasmic tail of the human CD28 T cell receptor engagement in the absence of CD28 cos- and binding partners. The human CD28 encodes a 220 amino acid-long timulation induces an unbalanced signaling response in which protein (218 in the mouse) that includes a leader sequence of 18 residues TCR-mediated Ca2+ influx predominates. This leads to activa- (19 residues in the mouse). The mature protein (202 residues) possesses a 41 amino acid-long cytoplasmic tail that includes three potential tion of calcineurin which dephosphorylates NF-AT leading to its protein-protein interaction motifs (highlighted in yellow). The nuclear translocation and induction of a limited set of anergy- phospho-Tyr173 within the YMNM motif serves as a docking site for the associated genes resulting in T cell anergy (Macian et al., 2004). SH2-containg proteins, p85, Grb2 and GADS. The P178 RRP motif can 190 CD28, in contrast to the TCR, does not induce a Ca2+ response interact with the SH3 domain of Itk. The P YAP motif can interacts with the SH3 domain of Grb2, GADS and Lck, as well as with filamin-A. (Lyakh et al., 1997). Instead, CD28-coupled costimulatory sig- Phosphorylation of Tyr191 within the PYAP motif creates a docking site for nals induce the activation of NF-κB and AP-1, and concomitant the Lck SH2 domain and enables PKCθ to interact via its V3 domain with AP-1 association with NF-AT, conditions that promote IL-2 prod the Lck SH3. Studies indicate that Tyr191 is important for CD28 and PKCθ localization to the cSMAC, and that the PYAP motif contributes to T cell uction and rescue of the T cells from a state of anergy (Macian activation and cytokine expression. et al., 2004).

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motif likely determine which of the three potential binding et al., 1993). More recent studies indicated that PKCθ can also partners interacts with the activated CD28 and, hence, the result- interact with the cytoplasmic tail of CD28, and that this inter- ing functional outcome. A second, nearby motif possesses the action involves Lck as an intermediate molecule, as discussed P178RRP sequence, and serves as a binding site for the SH3 below. domain of IL-2-inducible T cell kinase (Itk; Marengere et al., 1997; Garcon et al., 2004). CD28-mediated activation of Itk is CD28 AND THE IS dependent on Lck (Gibson et al., 1996), but the actual role of Itk Upon binding of its ligand, B7, CD28, similar to the engaged TCR, in CD28-induced costimulation is still controversial (Liao et al., accumulates at the cSMAC of the IS although the two receptors 1997; Gibson et al., 1998; Yang and Olive, 1999; Li and Berg, initiates distinct but complementary signaling pathways. The tran- 2005). A third, more distal, P190YAP motif serves as a poten- sient recruitment of CD28 to the immature IS of TCR engaged T tial docking site for several different effector molecules. These cells is very rapid and occurs within seconds of the onset of the include filamin-A, an actin binding protein and a scaffold for calcium signal (Andres et al., 2004). Engagement of the TCR in −/− lipid raft formation, which utilizes repeat 10 (amino acids 1158– Cd28 T cells results in altered, diffuse pattern of distribution 1246) for interaction with CD28 (Tavano et al., 2006), Grb2 of PKCθ and LFA-1 at the IS, suggesting an essential role for CD28 and GADS adaptor proteins, which bind the P190YAP motif via in the initiation and stabilization of the mature IS (Huang et al., their SH3 domain (Okkenhaug and Rottapel, 1998; Ellis et al., 2002; Sanchez-Lockhart et al., 2004). Furthermore, in vivo block- 2000), and the Lck and Fyn protein tyrosine kinases (PTKs; ing of CD28 impairs the activity of effector molecules, including Hutchcroft and Bierer, 1994; zur Hausen et al., 1997; Holdorf PKCθ (Jang et al., 2008), and inhibits T cell-dependent immune et al., 1999; Tavano et al., 2004). Both Lck and Fyn were impli- responses (Linsley and Nadler,2009). CD28 engagement promotes cated in the early phase of the CD28 signaling pathway (August a cytoskeleton-dependent recruitment of cell surface receptors et al., 1994) and coexpression studies demonstrated that the two (Wulfing and Davis, 1998) and signaling molecules-containing PTKs could phosphorylate CD28, primarily on Tyr173 at the lipid rafts that support building the IS and contribute to signal Y173MNM motif, thereby increasing the binding of p85- and transduction from IS-residing receptors (Dustin and Shaw, 1999; Grb2-SH2 to CD28 (Raab et al., 1995). Lck and Fyn were also Viola et al., 1999). found to coimmunoprecipitate with CD28 from activated T cells More recent studies demonstrated that in activated T cells, (Hutchcroft and Bierer, 1994), where Lck interacted with the CD28 is recruited coordinately with the TCR to form microclus- P190YAP motif via its SH3 domain (Holdorf et al., 1999; Tavano ters at the cSMAC (Yokosuka et al.,2008). Upon progression of this et al., 2004), and Fyn interacted with the same motif using its SH2 initial step, the CD28 and TCR segregate to two spatially distinct domain (zur Hausen et al., 1997), although other studies indi- subregions within the cSMAC, a central TCRhigh subregion, where cated no interaction between CD28 and Fyn (Marengere et al., signaling is terminated and TCR-associated signaling complexes 1997). While presence of the two proline residues in the P190YAP are internalized and degraded, and an outer TCRlow annular form motif predicts interaction with SH3-containg proteins, binding that contain CD28 clusters, as well as PKCθ. CD28 and PKCθ were studies demonstrated that the Lck-SH3 domain interacts with rel- physically associated, as shown by PKCθ coimmunoprecipitation atively low affinity (K d > 1 μM) with peptides that contain the with CD28 from a lysate of PMA-stimulated T cells (Yokosuka P190YAP motif and correspond to residues 188–202 of human et al., 2008). CD28, or 186–196 of murine CD28, respectively (Hofinger and Sticht, 2005). PKCθ–CD28 INTERACTION AND RECRUITMENT Other studies demonstrated that Tyr191 within the P190YAP OF PKCθ TO THE IS motif is one of two major phosphorylation sites in CD28- T cell receptor engagement polarizes PKCθ and induce its recruit- stimulated Jurkat T cells, and the only tyrosine residue within ment to the IS, a response that is greatly augmented by CD28 the CD28 cytoplasmic tail that is essential for delivery of cos- ligation (Huang et al., 2002; Tseng et al., 2008; Yokosuka et al., timulatory signals leading to CD69 expression and synthesis and 2008). Although the recruitment of PKCθ to the center of the secretion of IL-2 (Sadra et al., 1999). The latter findings raise IS (cSMAC) of is well documented, information on the molec- the possibility that CD28 engagement-induced phosphorylation ular basis for this highly selective localization has been relatively of Tyr191 creates a new and transient binding site for SH2- scarce. Early studies have shown that PKCθ recruitment to the containing proteins, possibly Lck, since CD28 and Lck were IS is indirectly dependent on the PI3K interaction motif within shown to colocalize at the cSMAC (Tavano et al., 2004; Kong the CD28 cytosolic tail (Harada et al., 2001). Thus, mutation et al., 2011). Binding studies provided further support for this of Met173 within the mouse YMNM motif, which binds PI3K hypothesis by showing that a CD28-derived peptide that pos- upon its tyrosine phosphorylation, resulted in decreased ability sesses phospho-Tyr191 interacts with the Lck-SH2 domain with of CD28 to direct PKCθ recruitment to the cSMAC, and inhib- a relatively high affinity (K d = 2.13 μM; Hofinger and Sticht, ited PKCθ-dependent activation of NF-κB to and the Il2 gene 2005), at the range of other SH2-ligand interactions (Bauer et al., (Sanchez-Lockhart et al., 2004). 2004). This binding affinity is about three orders of magnitude Following the recently reported PKCθ–CD28 association in stronger than that for the Lck-SH3 domain. High affinity bind- PMA-stimulated T cells (Yokosuka et al., 2008), we conducted a ing of Lck-SH2 to P190pYAP occurs despite the difference between detailed structure-function analysis of this association in TCR- this sequence and the phospho-YEEI sequence predicted to be stimulated T cells (Kong et al., 2011). We demonstrated that the preferred binding site of the Lck-SH2 domain (Songyang PKCθ physically associated with the cytoplasmic tail of CD28

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following TCR/CD28 costimulation. Taking advantage of the fact production, and Th2 and Th17 (but not Th1) differentiation and that PKCδ, the closest relative of PKCθ, does not translocate to inflammation. the IS after T cell-APC interaction (Monks et al., 1997), we com- Fine mapping of the PKCθ V3 domain identified an evolu- pared the amino acid sequence analysis of PKCθ and PKCδ and tionarily conserved proline-rich (PR) motif (ARPPCLPTP; corres- found that they diverged significantly only in their V3 (hinge) ponding to amino acid residues 328–336 of human PKCθ) within domain, corresponding to amino acids ∼291–378 of human PKCθ, the PKCθ-V3 domain, which was required for PKCθ–CD28 suggesting a potential role for this region in targeting PKCθ to association, PKCθ localization to the IS, and induction of PKCθ- the IS. Indeed, a V3-deletion mutant of PKCθ (PKCθ-ΔV3) or mediated functions. Insertion of this motif into the V3 domain of an exchange mutant of PKCθ, in which the native V3 domain PKCδ enabled this altered PKCδ form to translocate to the IS and was replaced by the PKCδ V3 domain, did not coimmunopre- activate PKCθ-dependent signal. The two internal proline residues cipitate with CD28, and failed to translocate to the IS (Kong in this motif (Pro-331 and -334) were particularly critical in this et al., 2011) and to activate PKCθ-dependent reporter genes such regard (Kong et al., 2011). as the CD28 response element (RE/AP). Conversely, the iso- In trying to more precisely define the nature of the inducible lated V3 domain of PKCθ localized in the center of the IS and PKCθ-Lck complex, we focused on the potential contribution of associated with CD28. Moreover, T cells recovered from mouse Lck kinase. This possibility was considered in view of previous BM chimeras on a Prkcq−/− background reconstituted with the studies demonstrating a functional relationship between CD28, same PKCθ mutants failed to proliferate and produce IL-2 in PKCθ, and Lck. First, in stimulated T cells, Lck can be recruited response to CD3/CD28 costimulation, and their ability to upreg- to the tyrosine-phosphorylated distal PR motif (P190Y*AP)inthe ulate CD69 or CD25 expression was reduced. Given the critical cytoplasmic tail of CD28 via its SH2 and SH3 domains, respectively role of the V3 domain in directing the CD28 association and IS (Miller et al., 2009;seeFigure 2), This motif directs the colocaliza- localization of PKCθ, we argued that this domain will function tion of PKCθ and CD28 to the cSMAC (Yokosuka et al., 2008) and as a dominant negative mutant by disrupting the activation- is apparently involved in additional biological functions, includ- dependent association between endogenous CD28 and PKCθ.As ing the stabilization of IL-2 mRNA, reorganization of lipid rafts, expected, ectopic expression of the isolated PKCθ V3 domain and sustained autophosphorylation and activation of Lck at the IS blocked the recruitment of endogenous PKCθ to CD28 and the (Holdorf et al., 2002; Sanchez-Lockhart et al., 2004; Dodson et al., IS, and severely inhibited PKCθ-dependent functions, includ- 2009). Second, Lck phosphorylates and associates with PKCθ, and ing CD25 and CD69 upregulation, T cell proliferation and IL-2 mutation of the major Lck phosphorylation site on PKCθ (Tyr90)

FIGURE 2 | A schematic model of the CD28-Lck-PKCθ tri-partite complex phospholipase C and hydrolysis of the membrane phospholipid (PIP2) formed inTCR/CD28-stimulatedT cells. TCR/ CD28 engagement triggers forms DAG, which enables PKCθ anchoring to the plasma membrane. the activation of tyrosine kinases and phosphorylation of multiple substrates, Colocalization of PKCθ and CD28 is regulated by an interaction between including the cytoplasmic tail of the CD28. Phosphorylation of a tyrosine the PKCθ PXXP motif and the Lck-SH3 domain, which results in the within the PYAP motif (Y191 in the mature human CD28) forms a high formation of a trimolecular complex comprising CD28-Lck-PKCθ. The inset afﬁnity-binding site for the SH2 domain of the Lck PTK. Lck is an IS-residing table shows the amino acid sequence of a region within the cytoplasmic molecule in activated T cells; it is tethered to the plasma membrane via its tail of the immature CD28 that includes the PYAP motif (on a yellow N-terminal palmitic and myristic fatty acids (Paige et al., 1993), and is background) compared to homologous sequences of three additional constitutively associated with the cytoplasmic tail of the IS-residing members of the CD28 coreceptor family (obtained using the ClustalW accessory molecules, CD4 or CD8 (Rudd et al., 1988, 1989; Veillette et al., multiple sequence alignment program). A partially conserved tyrosine is 1988; Barber et al., 1989; Paige et al., 1993). Simultaneous activation of marked in red.

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inhibited PKCθ-dependent activation events in stimulated T cells Despite the extensive amount of studies on the biology of (Liu et al., 2000). PKCθ in mouse T cells, very little is known about its regula- Our further analysis confirmed the physical and functional tion and function in human T cells. This is a substantial gap that CD28-Lck-PKCθ link by demonstrating that Lck function as an would need to be filled if PKCθ is destined to fulfill its promise intermediate to recruit PKCθ to CD28 upon T cell stimula- as a clinically relevant drug target (Altman and Kong, 2012). tion. The Lck-SH3 domain interacted with the PR motif in the As discussed earlier, the dependence of T cell-mediated delete- PKCθ V3 domain, while the Lck SH2 domain interacted with rious autoimmune/inflammatory responses, including GvHD, on phospho-Tyr191 in the P190YAP motif in the CD28 cytoplasmic tail. PKCθ, but its dispensable role in beneficial responses (antiviral Taken together, the above findings demonstrate a unique signaling immunity and GvL response) make it an attractive clinical drug mode of CD28 and establish the molecular basis for the spe- target with potentially advantage over global immunosuppressive cialized localization and function of PKCθ in antigen-stimulated drugs such as calcineurin inhibitors (e.g., cyclosporine A), which T cells. have pronounced toxic side effects. Indeed, there has been considerable interest among pharmaceutical companies in developing THE GLK-PKCθ LINK small molecule selective PKCθ catalytic activity inhibitors, and Recent studies demonstrated that recruitment of PKCθ to the AEB071,the most advanced of these compounds, which inhibits cSMAC in activated T cells is essential but not sufficient for the other PKC family members in addition to PKCθ, is currently in full activation of PKCθ and its downstream target molecules. These early clinical trials (Evenou et al., 2009). studies further showed that the germinal center kinase (GSK)-like Nevertheless, small molecule inhibitors of protein kinases often kinase (GLK) also translocates to the IS of TCR-engaged T cells have toxic side effects because of their lack of absolute speci- where it phosphorylates the activation loop of PKCθ,convert- ficity, which reflects the relatively high conservation of catalytic ing it into an active enzyme (Chuang et al., 2011). Of interest, domains within the protein kinase family, and even more so within however, despite the importance of PKCθ in the thymic devel- the PKC family. Furthermore, since catalytic kinase inhibitors in opment of natural regulatory T cells (nTregs; Schmidt-Supprian current clinical use are ATP competitors, they need to be used et al., 2004), GLK-deficient mice displayed normal nTreg develop- at relatively high and potentially toxic concentrations in order ment (Chuang et al., 2011). These results emphasize the important to effectively compete with ATP, whose intracellular concentra- role of post-transcriptional regulation of PKCθ that occurs at sev- tion is ∼1 mM. As a result, there has recently been considerable eral steps and involve different checkpoints at distinct sites within interest and progress in developing allosteric kinase inhibitors, the activated T cell. which bind to sites other than the catalytic site in kinases and, thus, are likely to be much more selective and less toxic (Lamba CONCLUSIONS AND FUTURE PERSPECTIVES and Ghosh, 2012). Our recent study (Kong et al., 2011) demon- Identification and characterization of the molecular mechanism strates a new potential approach for attenuating PKCθ-dependent by which PKCθ associates with CD28 and colocalizes with it at functions utilizing allosteric compounds based on the critical the cSMAC has provided important information relevant to the PR motif in the V3 domain of PKCθ that will block its Lck- mechanism by which CD28 and PKCθ contribute to signal trans- mediated association with CD28 and recruitment to the IS, which duction in TCR/CD28-engaged T cells. These findings also raise is obligatory for its downstream signaling functions. This new new questions relevant to the mechanism of interaction of CD28 approach could serve as a basis for the development of new and PKCθ and their specific role in the induction of distinct T therapeutic agents that would selectively suppress undesired T cell-mediated immune responses. One obvious question relates to cell-mediated inflammation and autoimmunity or prevent graft the mechanism by which PKCθ is sequestered away from the IS of rejection, while preserving desired immunity, such as antiviral activated Treg cells. It would be interesting to determine whether responses. a CD28-Lck-PKCθ tri-partite complex (Kong et al., 2011) occurs in Treg cells, and determine the mechanism that enables PKCθ ACKNOWLEDGMENTS recruitment away from the Treg-APC contact area. A possible This is manuscript number 1529 from the La Jolla Institute for explanation for this process was provided by Yokosuka et al. (2010) Allergy and Immunology. Work in our laboratory is supported by showing that CTLA-4 competes with CD28 in recruitment to the the USA-Israel Binational Science Foundation, the Israel Science cSMAC. In addition, it is not known whether PKCθ is involved Foundation administered by the Israel Academy of Science and in a second signal delivery during the costimulation of γδ Tcells Humanities, and by NIH grant CA35299. Noah Isakov holds the (Ribot et al., 2011). Joseph H. Krupp Chair in Cancer Immunobiology.

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Yokosuka, T., Sakata-Sogawa, K., feedback on regulatory T cell func- Conflict of Interest Statement: The Immun. 3:273. doi: 10.3389/fimmu. Kobayashi, W., Hiroshima, M., tion. Science 328, 372–376. authors declare that the research was 2012.00273 Hashimoto-Tane, A., Tokunaga, M., Zanin-Zhorov, A., Dustin, M. L., conducted in the absence of any com This article was submitted to Frontiers in Dustin, M. L., and Saito, T. (2005). and Blazar, B. R. (2011). PKC- mercial or financial relationships that T Cell Biology, a specialty of Frontiers in Newly generated T cell receptor theta function at the immunologi- could be construed as a potential con- Immunology. microclusters initiate and sustain T cal synapse: prospects for therapeu- flict of interest. Copyright © 2012 Isakov and Alt- cell activation by recruitment of tic targeting. Trends Immunol. 32, man. This is an open-access article dis- Zap70 and SLP-76. Nat Immunol 6, 358–363. tributed under the terms of the Creative 1253–1262. zur Hausen, J. D., Burn, P., and Received: 09 July 2012; accepted: 09 Commons Attribution License, which Zanin-Zhorov, A., Ding, Y., Kumari, S., Amrein, K. E. (1997). Co-localization August 2012; published online: 22 August permits use, distribution and reproduc- Attur, M., Hippen, K. L., Brown, M., of Fyn with CD3 complex, CD45 or 2012. tion in other forums, provided the origi- Blazar, B. R.,Abramson, S. B., Lafaille, CD28 depends on different mech- Citation: Isakov N and Altman A (2012) nal authors and source are credited and J. J., and Dustin, M. L. (2010). Pro- anisms. Eur. J. Immunol. 27, PKC-theta-mediated signal delivery from subject to any copyright notices concern- tein kinase C-theta mediates negative 2643–2649. the TCR/CD28 surface receptors. Front. ing any third-party graphics etc.

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